1. FIELD OF THE INVENTION
This invention relates to a novel plasmid and as novel microorganism containing the plasmid. The novel plasmid is useful for assaying promoter activity in Streptomyces as well as for increasing the yield of antibiotic fermentations.
2. DESCRIPTION OF THE PRIOR ART
In the prior art (Horinouchi, et al., J. Bacteriol. 162, 406-412 (1985)) the promoter-probe plasmid vector pARCl has been constructed. This plasmid vector has a unique BamHI cloning site that allows chromogenic identification of transcriptional control signals in Streptomyces lividans. Multi-copy promoter-probe plasmid vectors for Streptomyces have been described by Ward, et al. MGG 203 p.468-478 (1986) These promoter-probe plasmids may be used to isolate and characterize Streptomycetes promoters.
Horinouchi, et al. used the disclosed technique to isolate promoter containing fragments involved in the transcriptional regulation of gene expression. The disclosed plasmid allows for the direct detection of fragments involved in the transcriptional regulation of gene expression by the activation of brown pigment encoding genes in Streptomyces. In addition, promoters which function late in the life cycle can be readily detected. The significant shortcoming of the pARCl plasmid, in the analysis of gene expression signals, is the difficulty of reisolating the promoter containing fragments for subsequent analysis. Restriction enzyme sites are remote from the unique BamHI cloning site which makes it difficult to reisolate the cloned fragment. The basal level of brown pigment production in the intact vector is low and makes it difficult or impossible to detect low promoter activity.
Another class of prior art promoter-probe vectors utilizes the activation of a promoterless drug resistance gene. These methods are selective and require the early and constitutive expression of the cloned promoter in order to achieve significant levels of resistance. An example of a construct is PIJ486. Ward, et al. supra. This construct employs the aminoglycoside phosphorase transfer gene (aph) as a indicator system. This plasmid contains a transcriptional terminator originally isolated from phage fd in order to eliminate transcriptional readthrough from the vector into the aph gene. This plasmid also has a polylinker region containing the sites for several useful restriction enzymes.
The requirement for early and constitutive promoter activity precludes the detection of promoters which only express late in the life cycle of the organism.
The present invention comprises the construction of a novel plasmid that comprises a pARCl parent into which is inserted a transcriptional terminator and a polylinker sequence. This recombinant plasmid may be used to identify, isolate and characterize DNA fragments which contain functional promoters involved in the initiation and control of RNA synthesis. In addition, these DNA fragments are useful in making high yield Streptomyces strains for producing antibiotics. Examples of such products that are produced from Streptomyces are described in U.S. Pat. No. 4,468,462 which is incorporated by reference.
It has now been found that a Streptomyces plasmid, pCLL34 may be constructed from the previously disclosed plasmids pARCl and pIJ446.
Accordingly, it is a primary object of this invention to provide a Streptomyces promoter-probe vector for the analysis of transcriptional regulation.
It is also an object of this invention to provide a novel plasmid that may be utilized to increase the yield of an antibiotic from a Streptomyces such Streptomyces aureofaciens, Streptomyces noursei or Streptomyces aureus.